ANTI-HER2 AFFIBODY AND SWITCHABLE CHIMERIC ANTIGEN RECEPTOR USING SAME AS SWITCH MOLECULE

Abstract

Disclosed herein are an anti-HER2 affibody and a switch molecule including a cotinine-conjugated anti-HER2 affibody. When applied in combination with Cot-sCART, the cotinine-conjugated anti-HER2 affibody reacts with HER2-positive cells to induce immune cell activity, thereby finding advantageous applications as switch molecules in sCART therapeutic agents.

Claims

1. An anti-HER2 affibody, comprising the amino acid sequence represented by the following General Formula: TABLE-US-00009 General Formula VDNKFNKEX.sub.9X.sub.10X.sub.11AYWEIX.sub.17X.sub.18LPNLNX.sub.24X.sub.25QX.sub.27X.sub.28AFI X.sub.32X.sub.33LX.sub.35DDPSQSANLLAEAKKLNDAQAPK wherein, (i) X.sub.9X.sub.10X.sub.11 is LRV, X.sub.17X.sub.18 is VK, X.sub.24X.sub.25 is PY, PP, or PK, X.sub.27X.sub.28 is SR or IT, X.sub.32X.sub.33 is RS or KQ, and X.sub.35 is Y; (ii) X.sub.9X.sub.10X.sub.11 is LRG, X.sub.17X.sub.18 is TS, X.sub.24X.sub.25 is HS, X.sub.27X.sub.28 is IT, X.sub.32X.sub.33 is VS, and X.sub.35 is Y; (iii) X.sub.9X.sub.10X.sub.11 is MRD, X.sub.17X.sub.18 is VR, X.sub.24X.sub.25 is RI or PP, X.sub.27X.sub.28 is ST or SV, X.sub.32X.sub.33 is RS or RQ, X.sub.35 is Y; (iv) X.sub.9X.sub.10X.sub.11 is YML, X.sub.17X.sub.18 is VK, X.sub.24X.sub.25 is YP, X.sub.27X.sub.28 is QH, X.sub.32X.sub.33 is RS, and X.sub.35 is F; or (v) X.sub.9X.sub.10X.sub.11 is INK, X.sub.17X.sub.18 is IS, X.sub.24X.sub.25 is KE, X.sub.27X.sub.28 is HH, X.sub.32X.sub.33 is HS, and X.sub.35 is Y.

2. The anti-HER2 affibody of claim 1, wherein X.sub.35 is Y.

3. The anti-HER2 affibody of claim 1, wherein X.sub.10 is R.

4. The anti-HER2 affibody of claim 1, wherein X.sub.17 is V.

5. The anti-HER2 affibody of claim 1, wherein X.sub.24 is P.

6. The anti-HER2 affibody of claim 1, wherein the anti-HER2 affibody includes an amino acid sequence of any one of SEQ ID NOS: 1 to 8.

7. A switch molecule for activating chimeric antigen receptor-effector cells, the switch molecule comprising a cotinine-conjugated anti-HER2 affibody represented by the following General Formula: TABLE-US-00010 General Formula VDNKFNKEX.sub.9X.sub.10X.sub.11AYWEIX.sub.17X.sub.18LPNLNX.sub.24X.sub.25QX.sub.27X.sub.28AFI X.sub.32X.sub.33LX.sub.35DDPSQSANLLAEAKKLNDAQAPK wherein, (i) X.sub.9X.sub.10X.sub.11 is LRV, X.sub.17X.sub.18 is VK, X.sub.24X.sub.25 is PY, PP, or PK, X.sub.27X.sub.28 is SR or IT, X.sub.32X.sub.33 is RS or KQ, and X.sub.35 is Y; (ii) X.sub.9X.sub.10X.sub.11 is LRG, X.sub.17X.sub.18 is TS, X.sub.24X.sub.25 is HS, X.sub.27X.sub.28 is IT, X.sub.32X.sub.33 is VS, and X.sub.35 is Y; (iii) X.sub.9X.sub.10X.sub.11 is MRD, X.sub.17X.sub.18 is VR, X.sub.24X.sub.25 is RI or PP, X.sub.27X.sub.28 is ST or SV, X.sub.32X.sub.33 is RS or RQ, and X.sub.35 is Y; (iv) X.sub.9X.sub.10X.sub.11 is YML, X.sub.17X.sub.18 is VK, X.sub.24X.sub.25 is YP, X.sub.27X.sub.28 is QH, X.sub.32X.sub.33 is RS, and X.sub.35 is F; or (v) X.sub.9X.sub.10X.sub.11 is INK, X.sub.17X.sub.18 is IS, X.sub.24X.sub.25 is KE, X.sub.27X.sub.28 is HH, X.sub.32X.sub.33 is HS, and X.sub.35 is Y.

8. The switch molecule of claim 7, wherein the activation of the effector cell results in cytotoxicity against a target cell, cytokine secretion, or a combination thereof.

9. The switch molecule of claim 7, wherein the effector cell is selected from the group consisting of a dendritic cell, a killer dendritic cell, a mast cell, a natural killer cell, a B lymphocyte, a T lymphocyte, a macrophage, and a precursor cell thereof.

10. (canceled)

11. A switchable chimeric antigen receptor, comprising: (a) the switch molecule of claim 7; and (b) a chimeric antigen receptor comprising: i) an extracellular domain including an antibody targeting the switch molecule of claim 7, or an antigen binding fragment thereof; ii) a transmembrane domain; and iii) an intracellular signaling domain.

12. The switchable chimeric antigen receptor of claim 11, wherein the transmembrane domain includes a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T-cell receptor, CD27, CD28, CD3, epsilon, CD45, CD4, CD5, CD8 (CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

13. The switchable chimeric antigen receptor of claim 11, wherein the intracellular signaling domain includes a domain derived from a CD3ζ(CD3 zeta) chain.

14. The switchable chimeric antigen receptor of claim 11, wherein the intracellular signaling domain further comprises a costimulatory molecule selected from the group consisting of OX40 (CD134), CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), and 4-1 BB (CD137).

15. The switchable chimeric antigen receptor of claim 11, wherein the antibody of i) or the antigen binding fragment thereof is an anti-cotinine antibody.

16. The switchable chimeric antigen receptor of claim 11, wherein the antibody of i) or the antigen binding fragment thereof comprises HCDR1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, HCDR3 of SEQ ID NO: 11, LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0143] FIG. 1 is a graph showing affibodies specifically binding to HER2 in the form of periplasmic extracts.

[0144] FIGS. 2a, 2b and 2c show graphs illustrating binding affinity of HER2-binding affibodies for three HER2-expressing cells in terms of MFI values (A. NCI-N87, B. SK-OV-3, C. MDA-MB-231).

[0145] FIG. 3 is a graph quantitatively showing binding affinity of five Fc-coupled affibodies for HER2 protein.

[0146] FIGS. 4a, 4b, 4c and 4d show graphs quantitatively analyzing the binding affinity of five Fc-coupled affibodies for four HER2-expressing cell lines (A. NCI-N87, B. SK-OV-3, C. MDA-MB-453, and D. MDA-MB-231).

[0147] FIG. 5 is a graph quantitatively analyzing binding affinity of five cotinine-conjugated affibodies for HER2 protein.

[0148] FIG. 6 is a graph showing cytotoxicity effects of cotinine-conjugated affibodies and chimeric antigen receptor T cells (Cot-sCART) using and anti-cotinine antibodies on HER2-positive SK-OV-3 cells and HER2-negative Raji cells.

[0149] FIGS. 7a and 7b show effects of cotinine-conjugated affibodies and Cot-sCART on disease models constructed by applying SKOV3-Luc cells to immunodeficient mice (NSG) (A. Luminescence image, B. Luminescence signal quantitation data).

[0150] FIGS. 8a and 8b show effects of the cotinine-conjugated affibody and Cot-sCART on diseases constructed with SKOV3-Luc cells according to concentrations of the cotinine-conjugated affibody (A. Luminescence images, B. Luminescence signal quantitation data).

[0151] FIGS. 9a and 9b show quantitative comparisons of binding affinity of the affinity-improved clones produced in the Fc-coupled form for HER2 protein.

[0152] FIG. 10 is a graph showing quantitative analysis of the binding affinity of the cotinine-conjugated, optimized affibodies for HER2 protein.

[0153] FIG. 11 is a graph showing binding affinity of the cotinine-conjugated, optimized affibodies for the HER2-expressing cell line SK-OV-3.

[0154] FIG. 12 is a graph showing cytotoxic effects of the cotinine-conjugated, optimized affibody and the Cot-sCART on the HER2 positive cell line SK-OV-3.

DETAILED DESCRIPTION

[0155] Hereinafter, the present disclosure will be described in greater details with reference to the accompanying examples. However, these examples disclosed herein are only for illustrative purposes of the present disclosure, and it shall be obvious to a skilled person in the art that they should not be construed as limiting the scope of the present disclosure.

EXAMPLE

[0156] Unless stated otherwise, “%”, used to indicate concentrations of particular substances, stands for (wt./wt.) % for solid/solid, (wt./vol.) % for solid/liquid, and (vol./vol.) % for liquid/liquid throughout the specification.

Example 1: Development of Affibody to HER2

Example 1-1. Selection of Affibody by Panning

[0157] Clones binding specifically to HER2 were selected from an affibody library by panning with HER2-ECD-Fc protein. In addition, the clones were analyzed for binding to HER2 expression cells. As a result, five affibodies were obtained.

[0158] For use in panning, the affibody library was rescued in a phage form by using VSCM13 helper phage. As many as or more than 10.sup.13 library phages were initially allowed to bind the antigen, with a total of four panning rounds conducted. In the strategy of selecting phages exhibiting higher affinity, the amount of the antigen was decreased (10 μg, 5 μg, 2 μg, and 1 μg) while the number of washes was increased (3, 5, 7, and 10 time) as the panning round was further repeated.

[0159] Binder phages obtained in each panning round were analyzed for binding to the antigen. In this regard, ELISA was performed on the colonies resulting from infection into ER2537. In brief, a colony obtained by infecting binder phages was inoculated into an SB media (MOPS 10 g/L, Bacto YEAST extract 20 g/L, Trypton 30 g/L) and cultured until reaching an OD.sub.600 of 0.8, followed by shaking incubation at 30° C. in the presence of 1 mM IPTG (LPS solution, IPTG025) to allow the overexpression of the affibody. Periplasmic extraction was performed using a BBS buffer (200 mM Boric acid, 150 mM NaCl, 1 mM EDTA). Binders were screened by ELISA using the extract. For ELISA, the affibody periplasmic extract was applied to a plate coated with 2 μg/mL of each of HER1-ECD-Fc, HER2-ECD-Fc, HER3-ECD-Fc, and HER4-ECD-Fc and treated with a secondary antibody (anti-HA-HRP (Roche, 12013819001)). After color development with TMB (BioFX, TMBC-1000-01), OD.sub.450 values were read using an ELISA reader (PerkinElmer, Victor3) (FIG. 1). The ELISA data exhibited affibodies specifically binding to HER2 protein and 23 unique clones were identified by sequencing.

[0160] From the periplasmic extracts of the unique clones, cell binders were identified using the three HER2 expressing cell lines—NCI-N87 (ATCC, CRL-5822), SK-OV-3 (Korean Cell Line Bank, 30077), MDA-MB-231 (Korean Cell Line Bank, 30026). The three HER2 expressing cells were each prepared at a density of 5×10.sup.5 cells/tube and centrifuged at 1200 rpm for 3 min to harvest cells. The cells were washed with PBS containing 5% FBS and then incubated with 200 μL of the affibody-containing periplasmic extract at 4° C. for 1 hour. The cells were washed three times through three rounds of centrifugation with 200 μL of 5% FBS-containing PBS at 1200 rpm for 3 min. Then, the cells were incubated with 1 μg/mL anti-HA-FIT (Life Technologies, A11013) at 4° C. for 45 min in a light-shielded condition. After being washed three times through three rounds of centrifugation with 200 μL of 5% FBS-containing PBS at 1200 rpm for 3 min, the cells were measured for fluorescence intensity by FAGS (Beckmann coulter, FC500) (FIGS. 2a to 2c). Through the ELISA and cell binding test, five affibodies (ZQAA1, 7, 8, 11, 22) which had excellent binding affinity were selected (Table 1).

TABLE-US-00003 TABLE 1 Name Sequence SEQ ID NO: ZQAA1 VDNKFNKELRVAYWEIVKLPNLNPYQSRAFIRSLYDDPS 1 QSANLLAEAKKLNDAQAPK ZQAA7 VDNKFNKELRGAYWEITSLPNLNHSQITAFIVSLYDDPSQ 2 SANLLAEAKKLNDAQAPK ZQAA8 VDNKFNKEMRDAYWEIVRLPNLNPPQSTAFIRSLYDDPS 3 QSANLLAEAKKLNDAQAPK ZQAA11 VDNKFNKEYMLAYWEIVKLPNLNYPQQHAFIRSLFDDPS 4 QSANLLAEAKKLNDAQAPK ZQAA22 VDNKFNKEINKAYWEIISLPNLNKEQHHAFIHSLYDDPSQ 5 SANLLAEAKKLNDAQAPK

Example 1-2: Identification of Binding Affinity of Selected Affibody

[0161] The five selected affibodies were cloned in an Fc-conjugated form (Zb-Fc) and analyzed for binding affinity for HER2 protein and HER2 expressing cells. For ELISA, the five purified Zb-Fc forms were ⅕ diluted for 7 points starting from 60 nM in a plate coated with 2 μg/mL hHER2-ECD-His. After treatment with a secondary antibody (anti-hlgG-Fc-HRP (Invitrogen, H10007)), a color was developed with TMB. OD.sub.450 values were read using an ELISA reader, and EC.sub.50 values were measured by means of Graph prism (FIG. 3, Table 2).

TABLE-US-00004 TABLE 2 Affibody ZQAA1 ZQAA7 ZQAA8 ZQAA11 ZQAA22 EC.sub.50 (nM) 3.6 68.5 1.8 6.9 N/D

[0162] The affibodies were analyzed for binding affinity in HER2 expression cells. To this end, the four cell lines NCI-N87, SK-OV-3, MDA-MB-453 (Korean Cell Line Bank, 30131), and MDA-MB-231, which are in the descending order of HER2 expression, were each prepared at a density of 5×10.sup.5 cells/tube. The cells were harvested by centrifugation at 1200 rpm for 3 min and washed with PBS containing 5% FBS. Thereafter, the five Zb-Fc forms were ⅕ diluted for 7 points starting from 60 nM and incubated at 4° C. for 1 hour. The cells were washed three times by three rounds of centrifugation with 200 μL of 5% FBS-containing PBS at 1200 rpm for 3 min. Afterwards, the cells were incubated with 1 μg/mL anti-human-Fc-FITC (Life Technologies, A11013) at 4° C. for 45 min in a light-shielded condition. After being washed three times through three rounds of centrifugation with 200 μL of 5% FBS-containing PBS at 1200 rpm for 3 min, the cells were measured for fluorescence intensity by FAGS. From the MFI measurements, EC.sub.50 values were calculated using Graph prism (FIGS. 4a to 4d, Table 3).

TABLE-US-00005 TABLE 3 EC.sub.50 (nM) ZQAA1 ZQAA7 ZQAA8 ZQAA11 ZQAA22 NCI-N87 3.3 2.4 5.7 7.3 5.4 SK-OV3 2.7 1.9 6.2 14.8 8.3 MDA-MB-453 2.5 3.0 8.0 52.4 26.6 MDA-MB-231 2.3 7.1 5.6 N/D N/D

Example 2: Development of Cotinine-Conjugated Affibody and Identification of Activity Thereof

Example 2-1: Construction of Cotinine-Conjugated Affibody

[0163] Cotinine-conjugated affibody-cotinine complexes were synthesized such that the anti-HER2 affibodies of the present disclosure were used as switch molecules in an anti-switchable CAR system (Table 4).

TABLE-US-00006 TABLE 4 Name Sequence Cot-ZQAA1- trans-4-Cotinine carboxylic acid- Cot VDNKFNKELRVAYWEIVKLPNLNPYQSRAFIRSLYDDPSQSAN LLAEAKKLNDAQAPK-trans-4-Cotinine carboxylic acid Cot-ZQAA7- trans-4-Cotinine carboxylic acid- Cot VDNKFNKELRGAYWEITSLPNLNHSQITAFIVSLYDDPSQSANL LAEAKKLNDAQAPK-trans-4-Cotinine carboxylic acid Cot-ZQAA8- trans-4-Cotinine carboxylic acid- Cot VDNKFNKEMRDAYWEIVRLPNLNPPQSTAFIRSLYDDPSQSAN LLAEAKKLNDAQAPK-trans-4-Cotinine carboxylic acid Cot-ZQAA11- trans-4-Cotinine carboxylic acid- Cot VDNKFNKEYMLAYWEIVKLPNLNYPQQHAFIRSLFDDPSQSAN LLAEAKKLNDAQAPK-trans-4-Cotinine carboxylic acid Cot-ZQAA22- trans-4-Cotinine carboxylic acid- Cot VDNKFNKEINKAYWEIISLPNLNKEQHHAFIHSLYDDPSQSANL LAEAKKLNDAQAPK-trans-4-Cotinine carboxylic acid

Example 2-2: Identification of Binding Affinity of Cotinine-Conjugated Affibody for HER2

[0164] The five cotinine-conjugated affibodies constructed in Example 2-1 were measured for binding affinity for HER2 protein. First, ELISA was carried out, with HER2-ECD-His protein serving as an antigen. The cotinine-conjugated affibodies were each applied at a density of 2 μg/mL to HER2-ECD-His protein-coated plates. A secondary antibody (Anti-Cotinine IgG) was applied to the HER-ECD-His/cotinine-conjugated affibody complex and labeled with a tertiary antibody (anti-hlgG-Fc-HRP (Invitrogen, H10007)). The labeled complex was quantitated by developing a color with TMB and reading OD.sub.450 on an ELISA reader (PerkinElmer, Victor3) (FIG. 5).

Example 2-3. Construction of Lentivirus Containing Anti-Cotinine Antibody Fragment-Linked Chimeric Antigen Receptor

[0165] A chimeric antigen receptor was developed using an anti-cotinine antibody fragment. After being subjected to codon optimization for a CD8 leader, a scFv-type anti-cotinine, a CD8 hinge and transmembrane region, a CD137 cytoplasmic region, and a CD3 zeta cytoplasmic region in the chimeric antigen receptor, the gene was cut with Spel/Xhol and ligated to pLenti6-V5/DEST lentiviral vector (Invitrogen, V53306). The constructs thus obtained were identified by base sequencing. Amino acid and base sequences of the anti-cotinine antibodies and antigen-binding fragments thereof are represented in SEQ ID NOS: 9 to 18.

[0166] Each of the prepared lentiviral constructs was transduced, together with the plasmid pCMV-dR8.91 carrying the viral coat protein VSV-G (vesicular stomatitis Indiana virus G protein), gag, pol, and rev genes, into Lenti-X 293T cells (Takara Bio Inc., 632180). Transduction was performed using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer's protocol. After 72 hours, the cell culture containing lentivirus was 10-fold enriched by a centrifugal filter (Millipore, UFC910024) and stored.

Example 2-4. Preparation of Cytotoxic T Cell Presenting Anti-Cotinine Antibody-Bearing Chimeric Antigen Receptor on Surface (Cot-sCART)

[0167] Cytotoxic T cells on which anti-cotinine antibody fragment-bearing chimeric antigen receptors were presented were prepared using the lentivirus obtained in Example 2-3.

[0168] First, human naïve T cells were isolated and stimulated with Dynabeads™ Human T-Activator CD3/CD28 (Thermo Fisher Scientific, 11131D) for 24 hours. Thereafter, the lentivirus was transduced for 24 hours into the cells in the presence of polybrene (Sigma-Aldrich, H9268). Then, the medium was exchanged with a medium containing IL-2 (Gibco, CTP0021), followed by incubation at 37° C. in a 5% CO.sub.2 atmosphere. T cells presenting the anti-cotinine antibody fragment-bearing chimeric antigen receptor on the surface thereof (Anti-Cotinine CAR-T cell, Cot-sCART) were used in experiments within 24 hours after preparation.

Example 2-5. Identification of Cytotoxic Effect by Using Cotinine-Conjugated Affibody and Cot-sCART

[0169] The T cells Cot-sCART prepared in Example 2-4 and the cotinine-conjugated affibody complex were analyzed for introducing the activation of chimeric antigen receptor cells by recognizing HER2 on the cell surface.

[0170] Briefly, GFP-Luciferase-expressing lentivirus (Biosettia, GlowCell-16p-1) was introduced into the HER2-positive cell line SKOV-3 and the HER2-negative cell line Raji to establish SKOV3-Luc and Raji-Luc cell lines for use in experiments. First, SKOV3-Luc or Raji-Luc cells were seeded at a density of 1×10.sup.4 cells/well into 96-well plates. The prepared cytotoxic T cells were added to the Luc cells-seeded plate at a suitable rate per well. The cotinine-conjugated affibody was added at predetermined concentrations (0.1, 1, and 10 nM) to the test group treated with the Luc cells and the cytotoxic T cells and incubated 37° C. for 24 hours in a 5% CO.sub.2 atmosphere. Thereafter, cytotoxicity of the cytotoxic T cells was measured using a luciferase assay (Bio-Glo Luciferase assay system, Promega, G7941). For measurement of cytotoxic effects, the cytotoxic T cells, the cotinine-conjugated affibody, and the Luc cells were co-cultured after which the remaining SKOV3-Luc or Raji-Luc cells were lysed with 3X Lysis buffer (75 mM Tris(pH8.0), 30% glycerol, 3% Triton X100) to release a luciferase which was then reacted with a substrate. Relative lysis rates were given when the signal from the wells where only Luc cells had been cultured was set forth as 100%. As shown in FIG. 6, the cytotoxic effects were increased in a cotinine-conjugated affibody concentration-dependent manner. Relatively high cytotoxicity was observed in three cotinine-conjugated affibodies (Cot-ZQAA1-Cot, Cot-ZQAA8-Cot, and Cot-ZQAA11-Cot).

Example 2-6. Evaluation of Cotinine-Conjugated Affibody and Cot-sCART in Disease Animal Model

[0171] The chimeric antigen receptor cells were assayed for activity in disease animal models using the T cell Cot-sCART prepared in Example 2-4 and the cotinine-conjugated affibody complex.

[0172] Briefly, the HER2-positive SKOV3-Luc cell line established in Example 2-5 were intraperitoneally injected into immunodeficient NSG mice (The Jackson Laboratory, 005557). On day 5 after injection, luciferin (Promega, P1043) was intraperitoneally injected. Then, the SKOV3-Luc cells formed in the abdomen were measured and randomly grouped using a luminescence spectrometer (PerkinElmer, IVIS 100). On day 7, Cot-sCART (2.0E+6 CART cells) were peritoneally injected, followed by the cotinine-conjugated affibody (Cot-zHER2; 0.25 mg/kg) every two days. After peritoneal injection of the cotinine-conjugated affibody, SKOV3-Luc cells were monitored using a luminescence spectrometer. As can be seen in FIGS. 7a and 7b, the number of SKOV3-Luc cells was observed to be decreased only in the NSG mice to which the Cot-sCART and the cotinine-conjugated affibody were administered.

[0173] The activity of the chimeric antigen receptor cells was also monitored according to the concentration of the cotinine-conjugated affibody in disease animal models. Cot-sCART was peritoneally injected to the NSG mice where SKOV3-Luc cells had been established in the abdomen through the same peritoneal injection as in the aforementioned experiment. The NSG mice to which Cot-sCART was peritoneally injected were treated with 25 μg/kg and 2.5 μg/kg cotinine-conjugated affibody. As shown in FIGS. 8a and 8b, SKOV3-Luc cells were eliminated faster in the NSG mice to which the cotinine-conjugated affibody was injected at a higher dose.

Example 3: Affinity Maturation for ZQAA1 and ZQAA8

Example 3-1. Selection of Affinity-Improved Affibody Through Panning

[0174] Affinity maturation sub libraries of the affibodies ZQAA1 and ZQAA8 were constructed and selection was made of affibodies having improved affinity for HER2.

[0175] An affibody has a structure including three helices among which Helix1 and Helix2 are involved in binding a target and have variations on a total of 13 amino acid residues. For ZQAA1 and ZQAA8, sub-libraries in which random variations were given to 7 amino acid residues on Helix1 and 6 amino acid residues on Helix2 were constructed, respectively. Each sub-library was rescued in phage forms with VSCM13 helper phage before use in panning. As many as or more than 10.sup.13 library phages were initially applied to the antigen. Biotin-HER2-ECD-Fc was linked via Dynabeads M-280 streptavidin (Invitrogen, 11205D), followed by 5 rounds of panning in which the rescued phages were bound. In the strategy of selecting phages exhibiting higher affinity, the amount of the antigen was decreased (100 nm, 10 nM, 1 nM, 0.1 nM, and 0.01 nM) while the number of washes was increased (10, 15, 20, 25, and 30 times) as the panning round was further repeated. The binder phages obtained in each round of panning were analyzed for binding to the antibody as measured by ELISA for colonies resulting from infection into ER2537. Base sequencing identified 10 unique clones in the helix1 sub-library of ZQAA1, 23 unique clones in the helix2 sub-library of ZQAA1, 0 unique clones in the helix1 sub-library of ZQAA8, and 35 unique clones in the helix2 sub-library of ZQAA8.

[0176] Using periplasmic extracts of the unique clones, the two methods of extended wash and comparison of binding relative to expression were conducted to make selection against parental clones. Colonies resulting from infection of binder phages were inoculated into SB media (MOPS 10 g/L, Bacto YEAST extract 20 g/L, Trypton 30 g/L) and cultured until reaching an OD.sub.600 of 0.8, followed by shaking incubation at 30° C. in the presence of 1 mM IPTG (LPS solution, IPTG025) to allow the overexpression of the affibody. Periplasmic extraction of the affibodies was performed using a BBS buffer (200 mM Boric acid, 150 mM NaCl, 1 mM EDTA).

[0177] In the extended wash method, the affibody periplasmic extract was applied to the plate coated with 2 μg/mL HER2-ECD-Fc and then treated with a secondary antibody (anti-HA-HRP (Roche, 12013819001)). After color development with TMB (BioFX, TMBC-1000-01), OD.sub.450 values were read using an ELISA reader (PerkinElmer, Victor3). Simultaneously, the affibody periplasmic extracts were applied to the HER2-ECD-Fc-coated plates and stood in a wash buffer (0.05% Tween in PBS) for 2 hours, followed by treatment with a secondary antibody (anti-HA-HRP (Roche, 12013819001)). Color development with TMB (BioFX, TMBC-1000-01) was carried out before measurement of OD.sub.450 values by an ELISA reader (PerkinElmer, Victor3). Comparison of O.D. values was made between the plates that had stood and not stood in the wash buffer so as to select improved clones relative to parental clones.

[0178] In the method of comparing binding affinity relative to expression, a serial dilution of the affibody periplasmic extract from the original solution was applied to the plates coated with 2 μg/mL HER2-ECD-Fc and then treated with a secondary antibody (anti-HA-HRP (Roche, 12013819001)). After color development with TMB (BioFX, TMBC-1000-01), OD.sub.450 values were read on an ELISA reader (PerkinElmer, Victor3). In addition, a serial dilution of the periplasmic extract from the original solution was dotted to NC membranes. After the extracts were adsorbed and dried thereon, the NC membranes were incubated for 1 hour in 5% BSA in TBST. Thereafter, the membranes were treated with anti-HA-HRP (Roche, 12013819001) and then subjected to color development in an EDC solution (AbClon, Abc-3001). Expression levels were measured using an imaging system (Bio-rad, ChemiDoc touch). Selection was made of clones which exhibited ELISA OD.sub.450 values superior to the parental values at diluted concentrations at which expression levels were not detected. Five clones were selected for ZQAA1 and one clone for ZQAA8. Cloning was conducted in Zb-Fc forms.

[0179] Using ELISA and BLI methods, each of the Zb-Fc proteins produced in animal cells was measured for KD for HER2 protein in comparison with parental clones. For ELISA, the purified Zb-Fc proteins were each ⅕ diluted for 7 points starting from 60 nM before application to plates coated with 2 μg/mL hHER2-ECD-his protein. Treatment with a secondary antibody (anti-hlgG-Fc-HRP (Invitrogen, H10007)) was followed by color development with TMB (BioFX, TMBC-1000-01). OD.sub.450 values were measured using an ELISA reader (PerkinElmer, Victor3) (FIGS. 9a and 9b).

[0180] Some of the clones selected through ELISA were measured for KD values by a BLI method. Zb-Fc proteins were immobilized at a concentration of 2-5 μg/mL suitable for each clone to the AR2G sensor chips (PALL, 18-5092) by an amine coupling method using EDC/NHS. In order to measure KD values, ranging from 800 nM to 12.5 nM to the Zb-Fc-immobilized sensor chips, hHER2-ECD-his protein was allowed to bind at concentrations suitable for each clone for 10 min and then was dissociated for 15 min. As a result, clones which have KD values about 11-fold higher than those of the parental clones were acquired (Table 5). On the basis of the measurements, selection was made of ZQAA1234, ZQAA1239, and ZQAA8293 as affibodies optimized for HER2.

TABLE-US-00007 TABLE 5 Name Full R.sup.2 kon(1/Ms) kdis(1/s) KD(M) ZQAA1 0.98 2.5E+04 7.4E−03 2.9E−07 ZQAA1218 0.99 2.1E+04 3.1E−03 1.5E−07 ZQAA1233 0.99 2.4E+04 1.5E−03 6.5E−08 ZQAA1234 0.99 5.5E+04 1.4E−03 2.6E−08 ZQAA1239 0.98 3.3E+04 1.6E−03 4.9E−08 ZQAA1245 0.99 4.2E+04 5.5E−03 1.3E−07 ZQAA8 0.99 2.0E+04 5.0E−03 2.5E−07 ZQAA8293 0.99 4.0E+04 4.5E−03 1.1E−07

Example 4: Switch Production and Activity Analysis of Optimized Affibody to HER2

Example 4-1. Preparation of Cotinine-Conjugated Affibody

[0181] For use as a switch molecule in an anti-switchable CAR system, the optimized anti-HER2 affibody according to the present disclosure was synthesized into an antibody-cotinine complex (Switch molecule) through cotinine conjugation (Table 6).

TABLE-US-00008 TABLE 6 Name Sequence Cot-ZQAA1234 trans-4-Cotinine carboxylic acid- VDNKFNKELRVAYWEIVKLPNLNPPQITAFIRS LYDDPSQSANLLAEAKKLNDAQAPK Cot-ZQAA1239 trans-4-Cotinine carboxylic acid- VDNKFNKELRVAYWEIVKLPNLNPKQITAFIKQ LYDDPSQSANLLAEAKKLNDAQAPK Cot-ZQAA8293 trans-4-Cotinine carboxylic acid- VDNKFNKEMRDAYWEIVRLPNLNRIQSVAFIRQ LYDDPSQSANLLAEAKKLNDAQAPK

Example 4-2. Binding Affinity of Cotinine-Conjugated, Optimized Affibody for HER2

[0182] The three cotinine-conjugated, optimized affibodies prepared in Example 4-1 were analyzed for binding affinity for HER2.ECD. The binding affinity of the cotinine-conjugated, optimized affibodies for HER2.ECD was measured using ELISA in the same manner as in Example 2-2 (FIG. 10).

[0183] In addition, binding affinity for the HER2-positive cell line SKOV-3 was also measured. The cotinine-conjugated, optimized affibodies were labeled in the same manner as in Example 2-3 and the antibody fragments binding to the cell line were analyzed by flowcytometry (FIG. 11).

[0184] As can be seen in FIGS. 10 and 11, the three cotinine-conjugated, optimized affibodies (Cot-ZQAA1234, Cot-ZQAA1239, and Cot-ZQAA8293) were measured for binding affinity.

Example 4-3. Cytotoxic Effect of Cotinine-Conjugated, Optimized Affibody and Cot-sCART

[0185] The Cot-sCART prepared in Example 2-4 and the cotinine-conjugated, optimized affibody were analyzed for cytotoxicity. In this regard, an analysis was made to examine whether the T cell/cotinine-conjugated affibody complex could recognize HER2 on the cell surface to induce the activation of the chimeric antigen receptor cells. SKOV3-Luc or Raji-Luc cells were co-cultured with Cot-sCART in the presence of the cotinine-conjugated, optimized affibody in the same manner as in Example 2-5, after which the cytotoxic T cells were measured by a luciferase assay (Bio-Glo Luciferase assay system, Promega, G7941). As shown in FIG. 12, the cytotoxicity increased with the concentration of the cotinine-conjugated, optimized affibodies treated. Among the three cotinine-conjugated, optimized affibodies (Cot-ZQAA1234, Cot-ZQAA1239, and Cot-ZQAA8293), relatively high cytotoxicity was observed in Cot-ZQAA1234 and Cot-ZQAA1239.